Mesenchymal stem cells refer to primitive cells that are able to differentiate into bone, cartilage, adipose, nerve and muscle, for example, and are known as being contained in a large amount in bone marrow. In fact, mesenchymal stem cells are presently isolated from bone marrow and then studied for certain purposes or widely used in clinical trials for a variety of diseases.
Although it is easy to obtain mesenchymal stem cells from bone marrow, there are difficulty in acquisition of the bone marrow and further, difficulty to solve problems associated with an immunological rejection reaction occurring when implanting stem cells to others, at present.
Meanwhile, umbilical cord blood is relatively easy to obtain compared with bone marrow, and also, where great numbers of umbilical cord blood units are secured, it is possible to employ umbilical cord blood stem cells that are identical with or most similar to histocompatibility genes of patients and thereby it is possible to solve problems associated with immunological rejection. However, it is relatively difficult to obtain mesenchymal stem cells from umbilical cord blood, as compared to bone marrow and thereby there is difficulty in study and clinical applications.
Conventionally, as a method for isolating very minute amounts of mesenchymal stem cells contained in umbilical cord blood, there has been largely used a method involving separating a leucocyte layer using a density gradient centrifugation method and then culturing cells. However, it is likely to lose cells in the course of a density gradient centrifugation process, thus making it more difficult to culture mesenchymal stem cells that are present in umbilical cord blood in very minute amounts.
As conventional methods of isolating and culturing mesenchymal stem cells, reference may be made to U.S. Pat. Nos. 5,197,985 and 5,486,359, which disclose a method for proliferating mesenchymal stem cells in isolating and purifying mesenchymal stem cells from human bone marrow and cultivating them. That is, U.S. Pat. No. 5,197,985 is directed to a method for inducing human bone marrow-derived mesenchymal stem cells to differentiate into bone-forming cells, comprising: providing human bone marrow-derived mesenchymal stem cells that have been isolated, purified and culturally expanded from a bone marrow specimen by adding the bone marrow specimen to a medium which contains factors which stimulate mesenchymal cell growth without differentiation and allows, when cultured, for selective adherence of only the mesenchymal stem cells to a substrate surface; applying the isolated, purified and culturally expanded human bone marrow-derived mesenchymal stem cells to a porous carrier; and, implanting the porous carrier containing the culturally expanded human bone marrow-derived mesenchymal stem cells into an environment containing factors necessary for differentiating the human mesenchymal stem cells into bone cells, wherein said porous carrier comprises hydroxyapatite and tricalcium phosphate and wherein said medium is comprised of BGJb medium with fetal bovine serum (FBS) or is comprised of F-12 Nutrient Mixture. Further, U.S. Pat. No. 5,486,359 discloses isolated human mesenchymal stem cells which can differentiate into cells of more than one tissue type (for example, bone, cartilage, muscle or marrow stroma), a method for isolating, purifying and culturally expanding human mesenchymal stem cells, and characterization of and uses, particularly research reagent, diagnostic and therapeutical uses for such cells. In this patent, mesenchymal stem cells are derived from bone marrow and cultured in BGJb medium containing fetal bovine serum.